It is well documented in the orthopaedic literature that cigarette smoking has a negative impact on healing following long bone fracture and spinal fusion surgery. While the morbidity and financial burden of this clinical problem is significant, little progress has been made toward elucidating the underlying mechanisms that mediate the effects of smoking on skeletal healing. To begin addressing the question of mechanism, we propose to test a novel hypothesis which predicts that nicotine is a key component of cigarette smoke that affects the healing process by interacting with the nicotinic acetylcholine receptor (nACHR) expressed on mesenchymal stem cell membranes. More specifically, we hypothesize that activation of the nACHR in stem cells that are recruited to participate in healing processes that recapitulate endochondral ossification i) accelerates their progression through chondrogenesis and ii) inhibits or delays their subsequent progression to terminal maturation. To test this central hypothesis, we plan to address 3 Specific Aims. Experiments in Specific Aim 1 will characterize the impact of nicotine and cigarette smoke extract (CSE) on nACHR expression and signaling in mouse limb bud mesenchymal cells via mRNA and protein analysis and second messenger signaling assays. Experiments in Specific Aim 2 will compare the impact of nicotine and CSE on in vitro chondrogenesis and chondrocyte maturation in MSCs derived from wild type and alpha4 nACHR null mice. We will also determine the role of specific signaling events in the nicotine-evoked phynotypic changes seen in these cells. Lastly, in Specific Aim 3 we propose to compare the effects of nicotine and cigarette smoke on endochondral bone formation using the in vivo model of femur fracture healing in mice. Fractured wild type or alpha4 knockout mice exposed to nicotine or cigarette smoke will be analyzed via histomorphometric, in situ hybridization, microCT and biomechanical testing methods. Completion of these aims will for the first time evaluate the lynchpin role of nicotine as a key component in cigarette smoke that negatively influences skeletal healing. Furthermore, proposed experiments aimed at identifying the mechanism underlying the deleterious effects of nicotine during healing will pave the way toward the development of clinical strategies that alleviate the poor outcomes associated with smoking.